Confident assignment of site-specific glycosylation in complex glycoproteins in a single step.
Confident assignment of site-specific glycosylation in complex glycoproteins in a single step.
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DOI：
10.1021/pr500506z
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发表时间：
2014-10-03
影响因子：
4.4
通讯作者：
Zaia, Joseph
中科院分区：
文献类型：
--
作者：
Khatri, Kshitij;Staples, Gregory O.;Leymarie, Nancy;Leon, Deborah R.;Turiak, Lilla;Huang, Yu;Yip, Shun;Hu, Han;Heckendorf, Christian F.;Zaia, Joseph
糖蛋白可以含有几个糖基化位点，每个位点都是异质的。由于糖型多样性和非糖基化肽更有效地电离的信号抑制，典型的反相LC-MS和自下而上的蛋白质组学数据库搜索工作流程在识别复杂糖蛋白的位点特异性糖基化方面表现不佳。我们提出了一个LC-MS系统，用于在一个步骤中富集、分离和分析复杂糖蛋白（>4个N-糖基化序列）中的糖肽。该系统在反相C18-MS分析之前使用在线HILIC富集阱。我们证明了该系统的有效性，使用一组糖蛋白，包括人转铁蛋白（2序列），人α-1-酸性糖蛋白（5序列），和甲型流感病毒血凝素（9序列）。在线富集使糖肽成为检测到的最丰富的离子，从而促进产生高质量的数据依赖性串联质谱。串联质谱显示了使用碰撞活化解离检测的大多数糖肽的聚糖和肽骨架解离的产物离子，该解离可可靠地分配位点特异性糖基化。我们证明了我们的系统的价值，以确定位点特异性糖基化使用血凝素含有9个N-糖基化序列从一个单一的HILIC-C18-MS收购。
A glycoprotein may contain several sites of glycosylation, each of which is heterogeneous. As a consequence of glycoform diversity and signal suppression from nonglycosylated peptides that ionize more efficiently, typical reversed-phase LC–MS and bottom–up proteomics database searching workflows do not perform well for identification of site-specific glycosylation for complex glycoproteins. We present an LC–MS system for enrichment, separation, and analysis of glycopeptides from complex glycoproteins (>4 N-glycosylation sequons) in a single step. This system uses an online HILIC enrichment trap prior to reversed-phase C18-MS analysis. We demonstrated the effectiveness of the system using a set of glycoproteins including human transferrin (2 sequons), human alpha-1-acid glycoprotein (5 sequons), and influenza A virus hemagglutinin (9 sequons). The online enrichment renders glycopeptides the most abundant ions detected, thereby facilitating the generation of high-quality data-dependent tandem mass spectra. The tandem mass spectra exhibited product ions from both glycan and peptide backbone dissociation for a majority of the glycopeptides tested using collisionally activated dissociation that served to confidently assign site-specific glycosylation. We demonstrated the value of our system to define site-specific glycosylation using a hemagglutinin containing 9 N-glycosylation sequons from a single HILIC-C18-MS acquisition.
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影响因子：
7.4
作者：
Kirsch, Stephan;Zarei, Mostafa;Peter-Katalinle, Jasna
通讯作者：
Peter-Katalinle, Jasna
影响因子：
56.9
作者：
Ivens, AC;Peacock, CS;Myler, PJ
通讯作者：
Myler, PJ
影响因子：
7.4
作者：
Mysling, Simon;Palmisano, Giuseppe;Thaysen-Andersen, Morten
通讯作者：
Thaysen-Andersen, Morten
影响因子：
4.3
作者：
Thaysen-Andersen, Morten;Packer, Nicolle H.
通讯作者：
Packer, Nicolle H.
影响因子：
5.4
作者：
Abe, Y;Takashita, E;Hongo, S
通讯作者：
Hongo, S